| Wnt signaling plays key roles in metazoan embryonic development and in cancer. By expression cloning in Xenopus, I identified sizzled, a novel secreted wnt inhibitor resembling the extracellular domain of frizzled receptors but lacking their transmembrane segments. Expressed in a narrow ventral sector of the marginal zone and the prospective heart of early embryos, sizzled participates in patterning the ventral region of future mesoderm and restricts the ventral expansion of the territory fated to become muscle.;Wnt signaling controls β-catenin stability. I developed a novel Xenopus egg extract system that degrades β-catenin with kinetics identical to those observed in vivo. I demonstrated that axin, glycogen synthase kinase 3 (GSK3) and adenomatous poliposis coli protein (APC) are required for the ubiquitin-dependent β-catenin proteolysis. Axin dramatically accelerates β-catenin degradation and activates GSK3 by dephosphorylation. I reconstituted in vitro the inhibition of β-catenin turnover by dishevelled (dsh). Dsh binds axin and recruits the GSK3-binding protein (GBP) which competes with axin for binding to GSK3, an interaction required for β-catenin degradation. Dsh and GBP act synergistically to inhibit GSK3, therefore blocking β-catenin phosphorylation and destruction. Additionally, GSK3 inhibition reduces the phosphorylation of APC and its affinity for β-catenin, further decreasing β-catenin degradation.;β-catenin forms a transcriptional activator with Tcf3. Tcf3 blocks the degradation of β-catenin in extracts and embryos. Tcf3 competes with axin and APC for β-catenin, thereby sequestering β-catenin from its destruction machinery.;Axin is posttranslationally regulated by phosphorylation and ubiquitin-dependent proteolysis. Inhibition of GSK3 leads to axin dephosphorylation and rapid APC-dependent degradation. Thus both axin and β-catenin require APC for degradation but are inversely affected by GSK3 activity.;Our cell-free system for wnt signaling should help clarify elusive mechanistic aspects of the pathway. It should also allow the identification of small molecules that either inhibit or stimulate the pathway by affecting the stability of either β-catenin or axin. |
